GABAA and alpha-amino-3-hydroxy-5-methylsoxazole-4-propionate receptors are differentially affected by aging in the rat hippocampus

We have investigated the age-dependent modifications in the expression of eight different subunits of the gamma-aminobutyric acid, type A (GABA(A)) receptor (alpha1, alpha2, alpha3, alpha5, beta2, beta3, gamma2S, and gamma2L) and all four subunits of the alpha-amino-3-hydroxy-5-methylsoxazole-4-propionate (AMPA) receptor (GluR1-4) in the hippocampus of 24-month-old rats. All aged hippocampi displayed a remarkable increase (aged/adult ratio, 3.53 +/- 0.54) in the mRNA levels of the short version of the gamma2 subunit in parallel with a similar increase in the gamma2 subunit protein (aged/adult ratio, 2.90 +/- 0.62). However, this increase was not observed in the mature receptor. On the other hand, the expression of the different alpha subunit mRNAs increased moderately with aging, displaying a heterogeneous pattern. The most frequent modification consisted in an increase in the expression of the alpha1 subunit mRNA (aged/adult ratio, 1.26 +/- 0.18), in parallel with a similar increase on the alpha1 protein (aged/adult ratio, 1. 27 +/- 0.12) and in the alpha1 incorporated to the assembled GABA(A) receptor (tested by immunoprecipitation; aged/adult ratio, = 1.20 +/- 0.10). However, in the same hippocampal samples, no major modifications were observed on the expression of the AMPA receptor subunits. As a whole, these results indicated the existence of an increased expression of the GABA(A) receptor subunits and a preservation of the AMPA receptor at the hippocampal formation. These modifications could reflect the existence of specific deficiencies (neuronal loss and/or deafferentiation) on the GABAergic system in the aged rats.     

Role of T cell-replacing factor (TRF) in the murine B cell differentiation: induction of increased levels of expression of secreted type IgM mRNA

T cell-replacing factor (TRF) is known to play a critical role in the regulation of B cell growth and differentiation. In this study, the role of TRF in the expression of mRNA for both IgM and IgG1 class was investigated. The TRF was purified from cellfree supernatants from a T cell hybridoma, B151K12. RNA was isolated from chronic B cell leukemia (BCL1) cells, DNP-KLH-primed B cells, or normal B cells cultured with or without LPS, and LPS plus TRF or LPS plus BSF-1. The steady state level of isotype-specific mRNA was assessed by Northern blot analysis with a mu-specific or a gamma 1-specific probe. It was demonstrated that BCL1 and purified B cells cocultured with TRF expresses increased levels (twofold and fourfold, respectively) of secreted forms of mu mRNA. Purified B cells from DNP-KLH-primed mice also expressed increased levels (twofold to fourfold) of mu as well as gamma 1 mRNA for secreted form by stimulation with TRF. Total expression of mu mRNA, however, was approximately threefold higher than that of gamma 1 mRNA. The stimulation of normal B cells with LPS plus TRF induced an increase in the levels of mu mRNA and gamma 1 mRNA expression, fourfold and threefold, respectively. However, the levels of gamma 1 mRNA expression was one-third of that induced in B cells stimulated with LPS plus BSF-1. These results indicate that TRF preferentially induces increased levels of secreted type of mu mRNA and induces less gamma 1 mRNA than BSF-1. The differential role of TRF from BSF-1 in the expression of Ig mRNA will be discussed.     

"In vitro" effect of lipid peroxidation metabolites on elongation factor-2

Elongation Factor-2 (eEF-2) is the protein that catalyzes the translocation of the ribosome through mRNA. Not all oxidants affect eEF-2, which is extremely sensitive to oxidative stress caused mainly by lipid peroxidant compounds such as cumene hydroperoxide and t-butyl hydroperoxide. Lipid peroxides constitute a potential hazard to living organisms because of their direct reactivity with a variety of biomolecules and the ability to decompose into free radicals and reactive aldehydes. In this "in vitro" study, we show the effect of three of these aldehydes on the levels of hepatic eEF-2. The results suggest that the toxicity associated with prooxidant-mediated hepatic lipid peroxidation on protein synthesis can originate from the interaction of the aldehydic end products of lipid peroxidation with eEF-2.     

Radiation-induced alterations in rat mesangial cell Tgfb1 and Tgfb3 gene expression are not associated with altered secretion of active Tgfb isoforms

Despite evidence of selective radiation-induced modulation of expression of rat mesangial cell Tgfb gene isoforms, it is unclear whether these changes in gene expression are accompanied by changes in protein secretion. To address this issue, primary cultures of rat mesangial cells (passage number 6- 11) were placed in serum-free medium 24 h prior to irradiation with single doses of 0.5-20 Gy of (137)Cs gamma rays. After irradiation, cells were maintained in serum-free medium for a further 24 h. Irradiation of quiescent mesangial cells resulted in a significant (P 相似文献   

Up-regulation of expression of translation factors – a novel molecular mechanism for cadmium carcinogenesis

The molecular mechanisms potentially responsible for cadmium carcinogenesis were investigated by differential gene expression analysis of Balb/c-3T3 cells morphologically transformed with cadmium chloride. Differential display analysis of gene expression revealed overexpression of mouse Translation Initiation Factor 3 (TIF3; GenBank Accession Number AF 271072) and Translation Elongation Factor-1delta (TEF-1delta; GenBank Accession Number AF 304351) in the transformed cells compared with the control cells. The full length cDNAs for TIF3 and TEF-1delta were cloned and sequenced. Transfection of mammalian cells with an expression vector containing either TIF3 or TEF-1delta cDNA resulted in overexpression of the encoded protein. Overexpression of the cDNA-encoded TIF3 and TEF-1delta proteins in NIH3T3 cells was oncogenic as evidenced by the appearance of transformed foci capable of anchorage-independent growth on soft agar and tumorigenesis in nude mouse. Blocking the translation of TIF3 and TEF-1delta proteins using the corresponding antisense mRNA resulted in a significant reversal of the oncogenic potential of cadmium transformed Balb/c-3T3 cells as evidenced from the suppression of anchorage-independent growth on soft agar and diminished tumorigenesis in nude mouse. These findings demonstrate that the up-regulation of expression of TIF3 and TEF-1delta is a novel molecular mechanism responsible, at least in part, for cadmium carcinogenesis.     

应用实时定量RT-PCR技术检测b地中海贫血 珠蛋白基因的表达

为了定量检测 b 地中海贫血(b 地贫)的 a、b 和γ珠蛋白基因表达水平, 提取正常成人对照组、正常胎儿对照组和b 地贫患者组组成的样本 DNA,采用反向点杂交法（RDB）分析b 地贫各种突变类型;提取样本RNA用于进行针对a、b 和γ珠蛋白基因的荧光实时定量RT-PCR（FQ RT-PCR）。根据FQ RT-PCR原理,设计合成分别对应于a、b 和γ珠蛋白基因的3对引物和3条荧光探针,FQ RT-PCR在ABI 7700系统进行。用SPSS 10.0对实验数据进行统计学分析,分别计算正常对照组 (bA/bA,aa/aa),脐带血组(bA/bA,aa/aa),轻型b 地贫组(bT/bA,aa/aa),重型b地贫组(bT/bT,aa/aa)的a、b 和γmRNA比值,其中a/b分别为4.62±1.20、7.81±2.89、13.51±5.12、188.24±374.04;a/(b +γ)分别为4.43±1.17、0.56±0.49、9.62±4.37、2.14±1.58;γ/(b+γ) 分别为0.04±0.03、0.92±0.06、0.28±0.18、0.95±0.04。由于组与组之间均值变异范围较大,将其进行对数转换后再进行方差分析。结果表明： a/b与a/(b+γ)在所有组与组之间均有显著性差异。γ/(b+γ)除了在脐带血组和重型b地贫组之间无显著性差异外,在其他组与组之间均有显著性差异。实验说明,人类b珠蛋白基因的表达水平从正常对照组到重型b地贫组急剧下降且以重型b地贫组为最低;相反γ珠蛋白基因表达却明显升高,以重型b地贫组为最高。与正常成人对照组相比,胎儿期b mRNA水平较低但γmRNA 水平较高。因此,正常个体不同时期和不同类型b 地贫之间a、b与γ珠蛋白基因表达不同而且互相影响。 Abstract：whole blood samples were collected from 100 normal healthy adults, from umbilical cord of 33 newborn infants, 111 individuals with b-thalassemia minor (bT/bA,aa/aa) and 39 with b-thalassemia major (bT/bT,aa/aa). Prior to quantitative analysis of globin gene expression, DNA was extracted from all blood samples and used for b-thalassemia genotype analysis. Different types of b globin gene mutations were analyzed using reverse dot blotting (RDB) method. Total RNA were extracted and subjected to real-time RT-PCR for quantitative measurement of a, b andγglobin mRNA using three sets of primers and fluorescent-labeled probes, designed according to the sequences of a, b andγhuman globin gene. Real-time RT-PCR was performed in ABI 7700 system. Following the real-time RT-PCR, the mean values of a, b andγglobin mRNA were calculated and the ratios of a/b, a/(b + γ) andγ/(b + γ) were determined to characterize the relative expression levels of different globin genes among normal adult, infant, b-thalassemia minor and b-thalassemia major patients. The resultant data were analyzed using SPSS 10.0 software to determine statistical significance of human globin gene expression among normal controls and b-thalassemia patients. Due to vast variations of the mean globin gene mRNA levels among different groups, log conversion of a/b + 1, a/(b + γ) + 1 andγ/(b + γ) +1 was used for statistical analyses and intergroup comparison. The a/b globin gene mRNA ratios were determined to be 4.62±1.20, 7.81±2.89, 13.51±5.12, and 188.24±374.04 for normal healthy adult (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b-thalassemia major(bT/bT,aa/aa) respectively. The a/(b+γ) ratios were 4.43±1.17, 0.56±0.49, 9.62±4.37, and 2.14±1.58 for normal healthy adult (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b- thalassemia major(bT/bT,aa/aa) respectively. Theγ/(b+γ) ratios were 0.04±0.03, 0.92±0.06, 0.28±0.18, and 0.95±0.04 for normal healthy adult (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b- thalassemia major(bT/bT,aa/aa) respectively. Following statistical analyses, the a/b and a/(b+γ) globin gene mRNA ratios were significantly different among four different groups (normal adult, normal infant, b- thalassemia minor and b- thalassemia major). The γ/(b + γ) globin gene mRNA ratio was significantly different among all groups except for between infant and b- thalassemia major patients. Human b globin gene mRNA levels decrease progressively and dramatically from normal adults to b-thalassemia patients with b-thalassemia major having the lowest levels. On the other hand, the γglobin gene mRNA levels increase progressively from normal adult to b-thalassemia patients with b-thalassemia major having the highest levels. Infants have relatively lower levels of b but higher levels of γglobin gene mRNA as compared to those in normal adults. Thus, the relative expression levels of a, b or γglobin genes varied but inter-related among different ages of normal individuals and different b-thalassemia genotypes.     

ATP potentiates interleukin-1 beta-induced MMP-9 expression in mesangial cells via recruitment of the ELAV protein HuR

Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1 beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATP gamma S but conferred a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATP gamma S potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATP gamma S. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATP gamma S, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1 beta-inducibility. By confocal microscopy we demonstrate that ATP gamma S increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.     

5'-AMP-activated protein kinase activity and subunit expression in exercise-trained human skeletal muscle.

5'-AMP-activated protein kinase (AMPK) has been proposed to be a pivotal factor in cellular responses to both acute exercise and exercise training. To investigate whether protein levels and gene expression of catalytic (alpha(1), alpha(2)) and regulatory (beta(1), beta(2), gamma(1), gamma(2), gamma(3)) AMPK subunits and exercise-induced AMPK activity are influenced by exercise training status, muscle biopsies were obtained from seven endurance exercise-trained and seven sedentary young healthy men. The alpha(1)- and alpha(2)-AMPK mRNA contents in trained subjects were both 117 +/- 2% of that in sedentary subjects (not significant), whereas mRNA for gamma(3) was 61 +/- 1% of that in sedentary subjects (not significant). The level of alpha(1)-AMPK protein in trained subjects was 185 +/- 34% of that in sedentary subjects (P < 0.05), whereas the levels of the remaining subunits (alpha(2), beta(1), beta(2), gamma(1), gamma(2), gamma(3)) were similar in trained and sedentary subjects. At the end of 20 min of cycle exercise at 80% of peak O(2) uptake, the increase in phosphorylation of alpha-AMPK (Thr(172)) was blunted in the trained group (138 +/- 38% above rest) compared with the sedentary group (353 +/- 63% above rest) (P < 0.05). Acetyl CoA-carboxylase beta-phosphorylation (Ser(221)), which is a marker for in vivo AMPK activity, was increased by exercise in both groups but to a lower level in trained subjects (32 +/- 5 arbitrary units) than in sedentary controls (45 +/- 1 arbitrary units) (P < 0.01). In conclusion, trained human skeletal muscle has increased alpha(1)-AMPK protein levels and blunted AMPK activation during exercise.     

Role of glucose as a modulator of anabolic and catabolic gene expression in normal and osteoarthritic human chondrocytes

Cartilage matrix homeostasis involves a dynamic balance between numerous signals that modulate chondrocyte functions. This study aimed at elucidating the role of the extracellular glucose concentration in modulating anabolic and catabolic gene expression in normal and osteoarthritic (OA) human chondrocytes and its ability to modify the gene expression responses induced by pro-anabolic stimuli, namely Transforming Growth Factor-β (TGF). For this, we analyzed by real time RT-PCR the expression of articular cartilage matrix-specific and non-specific genes, namely collagen types II and I, respectively. The expression of the matrix metalloproteinases (MMPs)-1 and -13, which plays a major role in cartilage degradation in arthritic conditions, and of their tissue inhibitors (TIMP) was also measured. The results showed that exposure to high glucose (30 mM) increased the mRNA levels of both MMPs in OA chondrocytes, whereas in normal ones only MMP-1 increased. Collagen II mRNA was similarly increased in normal and OA chondrocytes, but the increase lasted longer in the later. Exposure to high glucose for 24 h prevented TGF-induced downregulation of MMP-13 gene expression in normal and OA chondrocytes, while the inhibitory effect of TGF on MMP-1 expression was only partially reduced. Other responses were not significantly modified. In conclusion, exposure of human chondrocytes to high glucose, as occurs in vivo in diabetes mellitus patients and in vitro for the production of engineered cartilage, favors the chondrocyte catabolic program. This may promote articular cartilage degradation, facilitating OA development and/or progression, as well as compromise the quality and consequent in vivo efficacy of tissue engineered cartilage.     

Expression profiling of the gamma-subunit isoforms of AMP-activated protein kinase suggests a major role for gamma3 in white skeletal muscle

Expression patterns of the three isoforms of the regulatory gamma-subunit of AMP-activated protein kinase (AMPK) were determined in various tissues from adult humans, mice, and rats, as well as in human primary muscle cells. Real-time PCR-based quantification of mRNA showed similar expression patterns in the three species and a good correlation with protein expression in mice and rats. The gamma3-isoform appeared highly specific to skeletal muscle, whereas gamma1 and gamma2 showed broad tissue distributions. Moreover, the proportion of white, type IIb fibers in the mouse and rat muscle samples, as indicated by real-time PCR quantification of Atp1b2 mRNA, showed a strong positive correlation with the expression of gamma3. In samples of white skeletal muscle, gamma3 clearly appeared to be the most abundant gamma-isoform. Differentiation of human primary muscle cells from myoblasts into multinucleated myotubes was accompanied by upregulation of gamma3 mRNA expression, whereas levels of gamma1 and gamma2 remained largely unchanged. However, even in these cultured myotubes, gamma2 was the most highly expressed isoform, indicating a considerable difference compared with adult skeletal muscle. Immunoblot analysis of mouse gastrocnemius and quadriceps muscle extracts precipitated with a gamma3-specific antibody showed that gamma3 was exclusively associated with the alpha2- and beta2-subunit isoforms. The observation that the AMPKgamma3 isoform is expressed primarily in white skeletal muscle, in which it is the predominant gamma-isoform, strongly suggests that gamma3 has a key role in this tissue.     

Semiquantitative detection of cytokine messages in X-irradiated and regenerating rat thymus

We investigated the expression of cytokine mRNA derived from thymocytes or thymic epithelial cells in X-irradiated (8 Gy) and recovering rat thymuses, according to our previous observation (Mizutani et al., Radiat. Res. 157, 281-289, 2002). The changes in mRNA expression level of interleukin 2 (Il2), Il4, tumor necrosis factor alpha (Tnf), interferon gamma (Ifng), and transforming growth factor beta (Tgfb) were examined. The mRNA expression of Il2 and Il4 decreased from day 5 to day 14 after irradiation. Thereafter, the expression level of Il2 mRNA recovered to normal control levels; however, the expression of Il4 mRNA tended toward significantly low levels. Tnf mRNA expression decreased on day 5 after irradiation and then showed a gradual increase back to normal control levels. Tgfb mRNA expression did not change significantly. Ifng mRNA expression was transiently enhanced from day 11 to day 14. The mRNA expression levels of Il10 increased significantly from day 3 to day 7 after irradiation. In addition, the mRNA expression of thymic epithelial cell-derived Il7 showed a transient decrease on day 3; however, then it showed a continuous increase from day 5 to day 21, finally reaching twice the normal control levels after X irradiation. These observations suggest that the expression of cytokine messages in the irradiated thymus changed significantly and did not return to normal for a long time after 8 Gy irradiation.     

ERCC1, XPD and RAI mRNA levels in lymphocytes are not associated with lung cancer risk in a prospective study of Danes

Low DNA-repair capacity has been associated with increased risk of several types of cancer. mRNA levels of the nucleotide excision repair genes ERCC1 and XPD have been shown to correlate with the DNA-repair capacity. Likewise, mRNA levels of several DNA-repair genes including ERCC1 have been shown to be lower in lymphocytes from patients with lung cancer and head and neck cancer compared with healthy persons. In these studies, the low DNA-repair gene expression levels could be either a risk factor for disease or a consequence of the same. In this nested case-cohort study, which to our knowledge, is the first prospective study of DNA-repair gene mRNA levels as predictors of lung cancer, we have investigated the occurrence of lung cancer in relation to the mRNA level of the two DNA-repair genes ERCC1 and XPD and the NF kappaB inhibitor RAI in blood samples prior to disease. Among 54,220 members of a Danish prospective cohort study, 265 lung cancer cases were identified and a sub-cohort comprising 272 individuals was used for comparison. The expression levels of the three adjacent genes were found to be highly inter-correlated, to be higher in women compared to men and to be lower in older individuals. The incidence rate ratios for lung cancer in association with one log-unit increase (natural logarithm) in mRNA levels were 1.12 (CI=0.89-1.41) for ERCC1, 1.00 (CI=0.83-1.21) for XPD and 1.25 (0.89-1.74) for RAI. In conclusion, this study indicated no association between mRNA expression of the DNA-repair genes ERCC1 and XPD and risk of subsequent development of lung cancer.